Thank you everyone for all of your responses to my questions concerning
EtOH precipitation. You've given us numerous leads to follow. I am
sending a clipping of some of the responses, and later I will report
what our success rate was.
OUr next question concerns the upgrade of the sequencers to the 48 and
64 well format. Ever cautious, I would like to know if anyone is is
using these upgrades successfully? We would be upgrading a stretch and a
non-stretch 373.
Here are your responses to the previous question, and Thanks again!
Naomi
Subject: to PPT or not to PPT
To: nobody at net.bio.NET
Message-id: <199607302125.OAA04880 at net.bio.net>
X-Envelope-to: thomson
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We have been using the FS enzyme for nearly a year, but have continued
to use
the centrisep columns for purification. Now, in order to cut costs and
to
prepare for future automation of the facility, we are considering
ethanol
precipitation.
There have been numerous communications concerning "T-blobs" and "loss
of an 80
base front" which have us concerned that the switch to EtOH ppt's will
decrease
the quality and reliability of the sequencing facility.
I would appreciate hearing the experience, good and bad, others in the
newsgroup
have had with this method. What protocols do you use? Has anyone managed
to
automate the dye-terminator reaction from A to Z.
Thanks for your input in advance.
Naomi Thomson
Thomson at BMS.com
(609) 252-5967MAIL>
From: IN%"hhills at iastate.EDU"
To: IN%"Thomson at bms.com"
CC:
Subj: Purification
Subject: Purification
X-Sender: hhills at pop-1.iastate.edu
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Naomi,
I get samples for gel runs that have been ethanol precipitated. They
are
not as clean as with the CentriSep columns. We are continuing to use
the
columns for our facility. Adjacent lanes are often affected when we run
the ppct samples on the same gel with our other samples.
Hal
Harold G. Hills, Ph.D., DNA Sequencing Specialist 515 294-9585
1184 Molecular Biology Building 515 294-1597 FAX
Iowa State University
hhills at iastate.edu
Ames, Iowa 50011-3260
#7 30-JUL-1996 19:36:21.77
NEWMAIL
From: IN%"ballard at fmppr.fmnh.ORG" "Bill Ballard"
To: IN%"nobody at net.bio.NET"
CC:
Subj: PPT or not to PPT
To: nobody at net.bio.NET
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X-Envelope-to: thomson
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Hey:
Here is a really simple protocol that we use at The Field Museum in
Chicago
for precipating 10=B5l cycle sequencing reactions-we do this in
96--plate
format. We don't get dye "blobs" and we don't loose the small fragments.
1. Add 40 =B5l ddH2O, 5 =B5l 3 M NaOAc, 125 =B5l Cold EtOH. Let
sit 1=
Hour
at -20=B0C (I am not sure how important this 1 hour at -20=B0C actually
is-=
-I
have used 30min at -20=B0C without a problem).
2. Spin for 15 min. Aspirate off the liquid.
3. Add 200=B5l 70% ethanol.
4. Spin for 5 min. Aspirate the liquid.
5. Dry in the speedvac.
Hope this helps:
Cheers
Bill Ballard
From: IN%"bishop at msvax.mssm.EDU" "David F. Bishop"
To: IN%"nobody at net.bio.NET"
CC:
Subj: RE: to PPT or not to PPT
MAIL>
#10 31-JUL-1996 06:01:57.18
NEWMAIL
Subject: Re: to PPT or not to PPT
To: nobody at net.bio.NET
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X-Envelope-to: thomson
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Dear Naomi,
We have used FS chemistry with CentriSep columns in the past with good
results as you find. We tried to switch to EtOH precipitation as
suggested
by ABI but were not satisfied with the results. The T-blob was present
as
were additional bands at the front from the incompletely removed dye
terminators. We also found the EtOH method to be more labor intensive
and
more subject to error - such as losing the pellet - than the desalting
column methods.
We now have worked out a method similar to the one Bruce Roe uses (see
his
web site for the full protocol: http://dna1.chem.uoknor.edu/) but with
PE
MicroAmp tubes. We punch a small hole in the bottom of the tube with a
needle, add a dab of glass beads and then an aliquot of Sephadex G-50
equilibrated in water. These minicolumns are placed in a PE microAmp
holder and the holder placed over another rack of MicroAmp tubes and
centrifuged in a microtiter plate rotor. The collection tubes are
SpeedVaced, reconstituted with loading buffer and transfered to the
plate
lanes. We get excellent recovery, no T-blobs and no residual dye
terinators.
As an additional note, we found that prewashing commercial columns with
water usually gave cleaner results than if used without prewash.
Cheers,
David
David F. Bishop, Ph.D. | EMail:bishop at msvax.mssm.edu
Dept. of Human Genetics, Box 1203 |
Mount Sinai School of Medicine | Phone: (212) 241-6946
5th Avenue and 100th Street, NY,NY 10029 | FAX: (212) 360-1809
MAIL>
#1 31-JUL-1996 18:57:21.51
NEWMAIL
Our experience with EtOH ppt has been less than pleasing and we have
reverted back to column purification. We didn$t have any problems with
T-Blobs but the first 50-60 bp were virtually unusable. If you can live
with this than go with the EtOH ppt if you$re doing only a few samples.
For a full run we have found that both methods take about the same
amount
of time and the extra usable data aquired with the column method is
worth
the extra expence.
Hope this helps :-)
Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada
Ph#: (613) 941-6441
FAX#: (613) 957-1358
E-mail: styler at hpb.hwc.ca